Journal of Neuroscience Research 55:629642 (1999)

Death of PC12 Cells and Hippocampal Neurons

Induced by Adenoviral-Mediated FAD Human
Amyloid Precursor Protein Gene Expression
Jin-Jun Luo,1* William Wallace,1 Teresa Riccioni,2 Donald K. Ingram,3
George S. Roth,3 and John W. Kusiak1
1Molecular Neurobiology Unit, Laboratory of Biological Chemistry, National Institute on Aging,
Baltimore, Maryland
2Laboratory of Cardiovascular Sciences, National Institute on Aging, Baltimore, Maryland
3Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, Maryland

We used adenoviral-mediated gene transfer of human

amyloid precursor proteins (h-APPs) to evaluate the
role of various h-APPs in causing neuronal cell death.
We were able to infect PC12 cells with very high
efficiency because D90% of the cells were cytochemically positive for -galactosidase activity when an
adenoviral vector containing LacZ cDNA was used to
infect cells. Cells infected with adenovirus containing
h-APP cDNA showed high-level transcription and
expression of h-APP as measured by reverse transcriptasepolymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17and 24-fold in cultures infected with recombinant
adenovirus containing wild-type mutant and 13- and
17-fold with V642F mutant. No elevation in h-APP
was seen in cultures infected with antisense h-APP or
null adenovirus. H-APP levels were maximal 3 days
after infection. Overexpression of V642F mutant hAPP in PC12 cells and hippocampal neurons resulted
in about a twofold increase in death compared with
overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral
mediated gene transfer in cell culture studies and
suggest that overexpression of a familial Alzheimers
disease mutant APP may be toxic to neuronal cells. J.
Neurosci. Res. 55:629642, 1999.
r 1999 Wiley-Liss, Inc.

Key words: Alzheimers disease; gene transfer; cell

culture; cell death; neurodegeneration
INTRODUCTION
Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by increasing loss of
memory and cognitive function, leading to dementia and
ultimately death. Pathological changes include the presence of senile plaques and neurofibrillary tangles distrib-

r 1999 Wiley-Liss, Inc.

uted in cortex and hippocampus and loss of neurons in

these areas. The main component of senile plaques is a
protein known as the amyloid beta protein (A; Mori et
al., 1992). A is derived from amyloid precursor protein
(APP; Kang et al., 1987; Haass et al., 1992), which is an
integral membrane protein (Dyrks et al., 1988). However,
the physiological and pathological roles of APP are not
completely understood (Selkoe, 1993; Mattson, 1997). In
general, it is believed that A plays a central role in AD
(Beyreuther et al., 1991; Bush et al., 1992; Selkoe, 1993,
1996; Iwatsubo et al., 1994; Price et al., 1994, 1995;
Iversen et al., 1995). In early-onset familial forms of AD
(FAD), several missense mutations have been found in
the APP gene. Overexpression of these mutant proteins
leads to increased amounts of A production or increased
amounts of longer forms of A (Citron et al., 1992, 1994;
Cai et al., 1993; Suzuki et al., 1994; Tamaoka et al., 1994;
Maruyama et al., 1996). The relationship between mutant
APP expression, A production, and neuronal death
observed in the brains of patients with FAD and the
mechanisms triggering the development of AD are still
under exploration (Yankner, 1992; Fukuchi et al., 1993;
Lannfelt et al., 1993; Cordell and Higgins, 1994; Hoyer,
1994; Frautschy et al., 1996; Gilman, 1996; Greenberg et
al., 1996; Selkoe, 1996; Hardy, 1997; Mattson, 1997).
Transgenic mouse models for AD have been developed, which display some of the changes in neurobiology,
histology, and behavior of AD (Yamaguchi et al., 1991;
Games et al., 1995; LaFerla et al., 1995; Hsiao et al.,
1996; Johnson-Wood et al., 1997; Sturchler-Pierrat et al.,

Contract grant sponsor: Intramural Program, NIA/NIH; Contract grant

sponsor: National Research Council, NIA/NIH.
*Correspondence to: Dr. Jin-Jun Luo, Mail Stop 12, MNU, LBC, GRC,
National Institute on Aging, National Institutes of Health, 5600 Nathan
Shock Drive, Baltimore, MD 21224. E-mail: luoj@vax.grc.nia.nih.gov
Received 1 September 1998; Revised 11 November 1998; Accepted 12
November 1998

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Luo et al.

1997; Price and Sisodia, 1998). However, detectable

evidence of AD-like pathology in these mice occurs
only after a period of at least 810 months. Moreover, even in the presence of A deposits, no clear
evidence of neuronal cell loss or neurofibrillary pathology has been reported in these models. Furthermore, in
these transgenic animals, the observed phenotypes may reflect
in part embryonic and postnatal effects of transgene expression because human APP (h-APP) overexpression occurs
throughout development in these animals.
Another approach to probe the biological functions
of APP is to use stable or transiently transfected cell lines
(Zhao et al., 1995, 1997; Yamatsuji et al., 1996). Overexpression of mutant APP induces apoptosis in these cell
lines (Wolozin et al., 1996; Yamatsuji et al., 1996; Zhao et
al., 1997). Recently we showed that expression of several
FAD mutant APPs induces apoptosis in pheochromocytoma (PC12) cells (Zhao et al., 1997) and the mutations in
the APP gene shift the protein processing toward the
amyloidogenic pathway in neuronal and endothelial cells
(Zhao et al., 1995). It is of more relevance to brain
functions to use primary neuronal cultures in these types
of studies. However, these cells, in general, are difficult to
transfect with high efficiency and are highly sensitive to
culture manipulations, whereas PC12 cells are well
characterized and can be differentiated into a neuronal
phenotype by neurotrophic factors such as nerve growth
factor (NGF). Much is known about their response to
toxic stimuli, and they are easily infected with adenovirus.
In the present report, we suggest that adenoviralmediated APP gene transfer can be used as an alternative
technique for the study of AD. The use of replication
defective adenovirus vectors as a means to introduce
exogenous genes into animals and cells is currently under
intensive investigation (Bajocchi et al., 1993; Becker et
al., 1994; Breakefield, 1993; Davidson et al., 1993, 1994;
Sallenave et al., 1998) and has proven to be useful in gene
therapy for some animal models of human diseases, such
as 1-antitrypsin deficiency (Rosenfeld et al., 1991) and
Parkinsons disease (Horellou et al., 1994; Ikari et al.,
1995; Choi-Lundberg et al., 1997; Horellou and Mallet,
1997). The methodology can target specific tissues (e.g.,
brains) and the timing of infection can be precisely
controlled in cell cultures or at different stages of
development in animals. Drawbacks to adenoviralmediated gene transfer include a limited time of gene
expression and nonspecific cellular toxicity at high-virus
titers. In the present study, we report that adenoviral
recombinants expressing wild-type or mutant APP genes
were efficiently introduced into PC12 cells and hippocampal cultures and that the expression of mutant APP caused
an increase in cell death in these cultures.

MATERIALS AND METHODS

Materials
The plasmids pCA4, pCA13, and pJM17 were gifts
from Dr. Jeffrey Chernak. APP751V717F and APP751V717I
cDNAs in pGEM3 were gifts from Dr. Boyu Zhao. Wild-type
(wt) APP695 cDNA in pGEM3 was a gift from Dr. Rachel
Neve. Anti-APP antibody (22C11) was purchased from
Boehringer Mannheim Corp. (Indianapolis, IN). Alpha-6
anti-APP antibody, which recognizes C-terminal amino acids
675695 of APP695, was a gift from Dr. Dale Schenk (Athena
Neurosciences Inc., South San Francisco, CA).

Culture Media
Medium A consisted of Dulbeccos Modified Eagles Medium (MEM) with 10% heat-inactivated horse
serum, 5% fetal bovine serum, 100 units/ml penicillin,
and 100 g/ml streptomycin. Medium B was the same as
medium A except that it contained 0.2% horse serum and
0.1% fetal bovine serum. Medium C consisted of improved MEM (IMEM) with 10% heat-inactivated fetal
bovine serum, 2 mM glutamine, 100 units/ml penicillin,
100 g/ml streptomycin, and 2.5 g/ml Fungizone. Medium D consisted of 1% agarose in MEM with 2 mM
glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 2.5 g/ml Fungizone. Medium E consisted of
Earls MEM with 10% heat-inactivated horse serum and
5% fetal bovine serum, 1 mM pyruvate, 100 units/ml
penicillin, and 100 g/ml streptomycin.

PC12 Cell Cultures

Rat pheochromocytoma (PC12) cells were maintained in medium A and plated at a density of 104
cells/8-chamber glass slide for -galactosidase cytochemistry, 2 105 cells/35-mm dish for Western immunoblot
assay, 13 104 cells/35-mm dish for cell counting, or
104 cells/well for 96-well plate for MTT assay; all dishes
were coated with collagen. After 1 day in medium A, the
cultures were washed once, and the medium was replaced
by medium B with or without recombinant adenovirus
(50 pfu/cell) expressing lacZ gene or different APP
cDNAs as described in detail in the text and cultured for
an additional 34 days unless otherwise noted.

Hippocampal Neuronal Cultures

Primary hippocampal cultures were prepared from
15-day-old Sprague-Dawley rat embryos. Under a dissecting microscope, the hippocampus was carefully dissected
out free of blood vessels in Ca/Mg-free Dulbeccos
phosphate-buffered saline (PBS). After incubation with
0.25% trypsin plus 1 mM EDTA in Ca/Mg-free Hanks
balanced salt solution for 20 min at 37C, the hippocam-

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631

pal tissues were washed once with medium E and then

dissociated into single cells by trituration in fresh medium E. The cell solution was incubated at room temperature for 10 min to allow the cell aggregates to settle. The
supernatant was transferred to a fresh tube, and the viable
cells were counted by trypan blue exclusion with a
hemocytometer. Single-cell preparations were plated on
poly-D-lysine- (20 g/ml) coated 6-well culture plates
(35 105 cells/1 ml medium/35-mm well). Cells in
medium E were maintained at 37C, 5% CO2:95% air for
34 hr before the medium was changed to fresh medium
E containing adenoviral recombinants, and the cultures
were continued for an additional 34 days.

7.8, containing 150 mM NaCl, 10 mM MgCl2, and 3%

sucrose). Virus was titrated via a plaque assay. The
individual recombinant adenoviral constructs containing
wt or mutant APP genes expressed in HEK293 cells were
verified by using RT-PCR to identify the appropriate
expression of h-APP mRNA or CMV fragments (data not
shown) and Western immunoblot assays to detect APP
expression (data not shown). Adenoviruses containing wt
APP cDNA in the sense orientation or in the antisense
orientation were named AdxS or AdxAS, respectively,
those containing mutant APP V642F, APP V642I, LacZ,
or adenoviral vector only were called AdxV642F,
AdxV642I, AdxLacZ, or AdxNull, respectively.

Construction of APP695 cDNAs

Plasmids containing APP751V717F or APP751V717I
(equivalent to V642F or V642I in APP695 isoform) were
digested with Bgl II/Spe I, and a 589-bp APP fragment
containing the V717F or V717I mutations was isolated by
agarose gel electrophoresis. These fragments were ligated
into a wt-APP695 cDNA construct, which was previously
digested with the same enzymes, to produce full-length
mutant APP695. These APP695 cDNAs were released from
pGEM3 by XbaI/HindIII digestion. The resulting 3.2-kb
APP695 fragments were inserted into the pCA shuttle
vectors previously digested with the same enzymes. All
constructs were verified by diagnostic restriction digestion and DNA sequencing.

Evaluating Dead Cells With Fluorescent

Sytox Staining
The cultures were evaluated by counting the number of dead and live cells in the cultures after the addition
of Sytox (Molecular Probes, Inc., Eugene, OR; final
concentration 1 M) at different time points. Sytox is
excluded by live cells but can pass through dead or dying
cell membranes and interact with nuclear DNA. The dead
cells, which were stained green, and live cells in the same
field were photographed with an inverted phase-contrast/
fluorescent microscope. The percentage of the dead cells
was calculated (200 cells/culture were counted). Statistical significance was determined by a two-tailed Students t-test.

Preparation of Recombinant Adenovirus

Homologous recombination was carried out by
cotransfection of HEK293 cells with calcium phosphate
method (Gibco/BRL, Gaithersburg, MD), with pJM17
and pCA4 or pCA13 vector (Graham et al., 1977; Graham
and Prevec, 1991) containing CMV promoter, driving
expression of wt or mutant APP695 cDNA. The cotransfected HEK293 cells were cultured in medium C overnight and then shifted to medium D. The plaques were
identified and isolated. The recombinants were expanded
several times on HEK293 cells until an adequate amount
of virus was achieved for purification. The cells (approximately 109 ) were suspended in Tris buffer (10 mM, pH
7.8), treated with 5% deoxycholate at 37C for 30 min,
followed by the addition of MgCl2 (1 M) and DNase (1
mg/ml) at 37C for an additional 30 min. After centrifugation, the supernatant was extracted three times with an
equal volume of 1,1,2-trichloro-1,2,2-trifluroethane before layering onto a double cesium chloride gradient
(specific gravities 1.4 and 1.2) and centrifugation at
70,000 g at 4C overnight. The viral band was removed
and diluted with an equal volume of Tris buffer (10 mM,
pH 7.8). This centrifugation procedure was repeated
twice. For storage, the virus was dialyzed four times
against 1 liter of storage buffer (10 mM Tris buffer at pH

-Galactosidase Cytochemistry and Histochemistry

Cultures were rinsed twice with PBS and fixed for 5
min in 4% paraformaldehyde plus 0.2% glutaraldehyde in
PBS. The cells were then washed twice with PBS
followed by incubation in a reaction mixture containing 1
mg/ml X-gal, 3 mM potassium ferricyanide, 3 mM
potassium ferrocyanide, and 1.3 mM MgCl2 in PBS at
37C for 24 hr.
RT-PCR
RT-PCR was used to distinguish endogenous rodent
and h-APP mRNA (Zhao et al., 1997). Total cellular RNA
was extracted directly from the cultured cells by using
RNAzoly B (Tel-Test, Inc., Friendswood, TX). RT-PCR
was carried out by using a cDNA Cycle kit (Invitrogen,
La Jolla, CA) according to the manufacturers instructions. After complementary DNA (cDNA) was synthesized, approximately one-sixth of the RT products was
used in a PCR amplification containing two sets of
primers for the same regions (809 bp) of rodent and
human APP mRNA. The sense primers annealed to
nucleotides 123, and the antisense primers were complementary to nucleotides 788809 of APP695. The cycle
number was 30 and the cycling profile was as follows: (1)
denaturation at 94C for 1 min, (2) annealing at 56C for

Fig. 1. Photomicrographs showing -galactosidase cytochemical staining in PC12 cells 3 days after infection with or without adenoviral constructs. Bar, 100 m.

Cell Death Induced by Mutant h-APP

633

Fig. 2. A strategy to detect the human amyloid precursor

protein (h-APP) gene in rodent (r-APP) cell lines by reverse
transcriptionpolymerase chain reaction (RT-PCR). Photograph
shows the RT-PCR products after ScaI digestion. Mk, Unt, S,

AS, and F denote molecular weight marker, uninfected cultures,

cultures infected with adenoviral recombinant carrying wildtype sense, antisense, or mutant V642F APP cDNAs, respectively; 1 and 2 indicate duplicate cultures.

1 min, and (3) extension at 72C for 2 min. The last cycle
had an additional extension at 72C for 10 min. The PCR
product from rodent APP mRNA contains a unique ScaI
restriction site (at 214 of rat APP695 sequence, Shivers et
al. 1988) and can be digested into two smaller fragments
(214 and 595 bp), whereas the product from h-APP
mRNA remains undigested (809 bp).

Detection of Human APP Messenger

RNA in PC12 Cells
Using RT-PCR, h-APP mRNA was detected in cells
infected with adenovirus containing either wt or mutant
APP cDNAs (Fig. 2). Uninfected naive cultures or
cultures infected with a null adenovirus (AdxNull) contained endogenous APP mRNA but no detectable h-APP.
A weak 800-bp band was detected in the cultures infected
with adenovirus containing antisense APP cDNA. This
weak band may derive from the PCR amplification of the
antisense adenovirus APP construct. These results indicate that human APP cDNAs can be introduced into PC12
cells by adenovirus and transcribed.

Western Immunoblot Assay

Assays were carried out by using enhanced chemiluminescence as previously described (Wallace et al.,
1995). Densitometric quantitation was performed with
NIH Image software.

RESULTS
Introduction of -Galactosidase Into PC12 Cells
We first tested the efficiency of adenoviral-mediated
gene transfer in PC12 cells. Three days after infection
with adenovirus carrying LacZ cDNA (AdxLacZ), -galactosidase activity was detected in the cell nuclei,
cytoplasm, and processes of PC12 cells (Fig. 1). The
efficiency of the infection with adenovirus was 8090%
under our culture conditions. These data demonstrate that
adenovirus-mediated gene transfer is highly efficient in
PC12 cells.

Expression of APP in PC12 Cells

To determine the optimal conditions for the expression of h-APP introduced by adenovirus in PC12 cells, we
carried out time-course and dose-response experiments.
PC12 cells were plated as described and infected with 10
pfu/cell of adenovirus containing either sense (AdxS) or
anti-sense (AdxAS) wt APP cDNAs or AdxNull for
14 days. The conditioned medium from each infection was subjected to Western immunoblot assay and
subsequently quantitated by using NIH image analysis.
As shown in Figure 3A, very low levels of APP immunoreactivity were seen in the cultures treated with
AdxNull or AdxAS for 14 days, whereas a large in-

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Fig. 3. Expression of amyloid precursor protein (APP) in PC12

cell cultures measured by Western immunoblot assay and
densitometric quantitation. A: Time course of APP expression
in PC12 cells infected with adenoviral recombinants (10
pfu/cell). Ordinate is the relative density of APP immunoreactivity. Results represent the mean SEM from a pool of two sets
of experiments, each containing duplicate cultures; error bars
are not shown when the SEM value was less than the symbol.

B: Dose-response curve for APP expression in PC12 cells.

Ordinate is the relative density of APP immunoreactivity. Xs,
diamonds, and circles represent cultures infected with wild-type
adenoviral vector, adenoviral recombinant carrying wild-type
antisense, and sense human APP cDNAs, respectively. Results
are the mean SEM of two individual experiments; error bars
are not shown when the value was less than the symbol.

crease in APP levels was found in the AdxS-treated

cultures. Infecting the cultures with AdxS for 3 days
resulted in an 14-fold increase of APP levels in the
medium as compared with that from the medium of
control cultures treated with AdxAS or AdxNull virus.
The level of APP expression induced by AdxS was
dose dependent. A concentration of AdxS at 50 pfu/cell
induced maximal APP expression. Concentrations higher than
50 pfu/cell resulted in a significant cell death, probably
resulting from the nonspecific toxicity of adenovirus (data not
shown). In contrast, infection with increasing amount of
AdxAS or AdxNull adenovirus did not increase APP expression (Fig. 3B). The optimal conditions to induce maximal
expression of APP in PC12 cells were infecting cultures with
AdxS at a concentration of 50 pfu/cell for 3 days.

AdxNull or AdxAS (Fig. 4A). Secreted APP levels were

increased 24fold in the cultures infected with AdxS
and 17-fold in cultures infected with AdxV642F compared with AdxNulltreated cultures. The intracellular
expression of APP appeared in a pattern similar to that
observed for extracellular APP (Fig. 4B). The intracellular APP level was increased 17-fold in cultures infected
with AdxS and 13-fold with AdxV642F. The introduced
h-APP appeared to be processed and secreted similarly to
endogenous protein in that antibody alpha-6, recognizing
COOH-terminal epitopes of APP, detected APP intracellularly but not extracellularly. In contrast, antibody 22C11,
recognizing an NH2-terminal region of APP, detected
APP both extracellularly and intracellularly (Fig. 5).
Thus, the overexpression of h-APP did not interfere with
the endogenous secretase processing of APP. Increased
amounts of intracellular COOH-terminal fragments
(Fig. 5) were detected by alpha-6 antibody in AdxS- and
AdxV642F-infected cells. Based on electrophoretic mobility, these fragments appear to be COOH-terminal

Intracellular Expression and Extracellular

Secretion of APP in PC12 Cells
Very low levels of secreted APP were observed in
control culture media or media of cells infected with

Cell Death Induced by Mutant h-APP

635

Fig. 4. Extracellular (A) and intracellular (B) levels of amyloid

precursor protein (APP) in PC12 cell cultures measured by
Western immunoblot assay and densitometric quantitation. Unt,
Null, AS, S, and V642F are the uninfected cultures, the cultures
infected with wild-type adenovirus, adenoviral recombinant
containing wild-type antisense, wild-type sense, and mutant

V642F APP cDNAs, respectively. The ordinate is the fold

increase in APP immunoreactivity relative to null. Bars represent the mean SEM from a pool of two sets of experiments,
each containing duplicate cultures; error bars are not shown
when the value was less than the symbol.

fragments (CTF) generated by and secretase activity.

These data demonstrate that adenoviral transfer of
h-APP cDNAs into PC12 cells led to their increased
intracellular expression, processing, and extracellular
secretion.

The percentage of dead cells was not significantly

different in uninfected cultures (14.1 2.7%) or cultures infected with AdxNull (13.4 2.8%), AdxS
(16.4 2.0%), and AdxAS (11.7 3.0%). These results are consistent with those of previous reports on
increased apoptosis in stable transfected PC12 cell
lines (Zhao et al., 1997) and COS cells (Yamatsuji et al.,
1996) expressing mutant APPs.

PC12 Cell Death Induced by Adenoviral-Mediated

APP Gene Transfer
Recent reports have shown that expression of
mutant APP induces apoptosis in neuronal-like PC12
cells (Zhao et al., 1997) and nonneuronal COS cells
(Yamatsuji et al., 1996). We employed Sytox dye labeling and cell counting techniques to evaluate survival
of PC12 cells infected with adenoviral constructs.
Shrunken cell somata with condensed nuclei were
labeled with Sytox, suggesting apoptotic morphological changes (Fig. 6). Figure 7 summarizes the results
from eight individual experiments of PC12 cells. A
significant increase in the number of dead cells was
observed in the cultures infected with AdxV642F
(26.2 4.0%, P 0.05 vs. AdxNull-treated cultures).

Primary Hippocampal Neuronal Cell Death Induced

by Adenoviral-Mediated APP Gene Transfer
We also tested whether adenovirus-expressing
APP cDNAs could cause cell death in cultured primary
hippocampal neurons. Cultures were infected with 1020
pfu/cell adenoviral constructs and examined after 5 days
using Sytox fluorescent nuclear labeling (Fig. 8). We
observed shrunken cell somata, with condensed nuclear
chromatin labeled by Sytox, suggesting an apoptotic cell
death. Quantitation of cell death from nine individual
experiments was performed (Fig. 9). A significant in-

Fig. 5. Extracellular and intracellular levels of amyloid precursor protein (APP) in PC12 cell cultures measured by Western
immunoblot assay using N-terminal antibody (22C11) and
COOH-terminal antibody (alpha-6). The upper panel shows
the results with antibody 22C11 and the lower with alpha-6.

Wt-hAPP, V642I and V642F represent the cultures treated with

adenoviral recombinant containing either wild-type sense,
mutant V642I, or mutant V642F APP cDNAs, respectively.
CTF, COOH-terminal fragment.

Cell Death Induced by Mutant h-APP

637

Fig. 6. Fluorescent Sytox dye analysis of PC12 cells treated

with different adenoviral constructs. Cultures were prepared as
described in Materials and Methods and were infected with
vehicle (A and B), wild-type adenoviral vector (C and D),
adenoviral recombinant carrying wild-type antisense (E and F),

wild-type sense (G and H) and mutant V642F APP (I and J)

cDNAs, respectively. A, C, E, G, and I are phase-contrast
images of the exact same fields shown in B, D, F, H, and J under
fluorescence. Bar 40 m.

crease in the number of dead cells was found in the

cultures infected with AdxS and AdxV642F (47.7 4.3
and 46.32 1.95, respectively; P 0.005) vs. AdxNull
or uninfected cultures (27.3 5.1 or 24.4 4.1). Interestingly, an increase in the number of the dead cells was also
observed in the cultures infected with AdxAS (36.8 5.1),
which reached a significant level (P 0.05).

DISCUSSION
Our studies demonstrate that adenoviral recombinants carrying h-APP genes can efficiently infect PC12
cells and that overexpression of FAD mutant APP genes
induces cell death. Our studies also suggest adenoviralmediated gene transfer may be used as an alternative

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Luo et al.

Fig. 7. Percentage of dead cells in PC12 cell cultures infected

with different adenoviral constructs using Sytox exclusion
analysis. Unt, Null, AS, S, and V642F represent the uninfected
cultures, the cultures infected with wild-type adenoviral vector
or adenoviral recombinant containing wild-type antisense,
sense, or mutant V642F APP cDNAs, respectively. Results are
the mean (SEM) of eight separate experiments, each containing at least three individual evaluations. Asterisk represents a
statistically significant difference from null cultures (P 0.05).

technique in the creation of cell culture models for studies

on AD and will be useful in the study of the pathogenesis
of the disease.
The infection efficiency of PC12 cells was very
high because 80% were positive for -galactosidase
cytochemical staining after infecting cultures with AdxLacZ (Fig. 1). RT-PCR analysis, which distinguished
exogenous h-APP mRNA from endogenous rodent APP
mRNA (Fig. 2), showed that the h-APP cDNA could be
efficiently transcribed in the host cells. The extracellular
secreted levels of h-APP were increased 24-fold over
endogenous levels in cultures treated with AdxS and
17-fold with AdxV642F. The amount of intracellular APP
and secreted APP is slightly greater in cells infected with
AdxS, which may be due to slight differences in the
infectibility or purity of the viral preparations. Nonetheless, this result suggests that expression of the mutant
form of APP may actually be slightly more toxic than the
results suggest. Western blots using alpha-6 antibody,
which recognizes the COOH-terminal region of APP, also
showed increased amounts of intracellular full-length
APP. The processing of h-APP appears to be similar to
endogenous APP. Two protein bands migrating at 10
kDa were detected in the cell lysates from the cultures
infected with adenovirus containing h-APP cDNA (Fig.
5) but not in lysates of naive cells or cells infected with
AdxNull or AdxAS (data not shown). These double bands

may include CT-100, which is a potentially neurotoxic

peptide (Neve et al., 1996; Oster-Granite et al., 1996;
McPhie et al., 1997). Interestingly, higher levels of CTFs
were found in the cell lysates from the cultures infected
with adenovirus expressing mutant h-APP cDNAs than in
wt h-APP cell lysates, suggesting that expression of
mutant forms of APP may lead to production of more
intracellular amyloidogenic APP fragments. It will be
important to determine intracellular levels of A peptide
in these infected cultures.
The levels of APP expression induced by adenoviralmediated APP gene transfer are much higher than those
we previously observed in stable transfected cell lines
that required NGF and cAMP to promote APP expression
(Zhao et al., 1995, 1997). In the presence of NGF and
cAMP, secreted APP levels were increased about six- to
eightfold compared with controls in both the wt and
V642F stable transfected cell lines. Very low levels of
h-APP expression were detected in these stable transfected cell lines when the cells were not treated with
growth factors (Zhao et al., 1995). Thus, adenoviralmediated APP gene transfer has a clear advantage in APP
expression in PC12 cells over the transfected stable cell
lines because high-level expression is observed in the
absence of growth factors. Obviating the need for growth
factors (Refolo et al., 1989; Quon et al., 1990; Clarris et
al., 1994; Haring et al., 1995; Lahiri and Nall, 1995;
Cosgaya et al., 1996; Schmitt et al., 1996; Slack et al.,
1997) may simplify the experimental models (Butcher
and Woolf, 1989; Yankner et al., 1990; Frade et al., 1996;
Carlson et al., 1997).
The results from the present study using adenoviralmediated gene transfer in PC12 cells and in primary
hippocampal neurons support the hypothesis that the
expression of mutant APPs can induce cell death. This
observation is in agreement with previous reports that the
overexpression of mutant APPs in stably transfected COS
and PC12 cells result in apoptosis (Yamatsuji et al., 1996;
Zhao et al., 1997). An adenoviral recombinant carrying
mutant V642I h-APP gene also significantly promoted
cell death in PC12 cells (data not shown).
In addition to the increased number of dead cells
induced by AdxV642F, the number of dead cells was also
elevated in primary hippocampal cultures overexpressing
wt h-APP (Fig. 9). To explain this discrepancy between
the results from the cultures of PC12 cells and primary
hippocampal neurons, we have to compare the properties
of these different cells. PC12 cells are from an immortalized cell line that has the capacity to grow indefinitely in
culture and, therefore, their properties are clearly different from those of primary hippocampal neurons, which
do not have the ability to proliferate. Cell lines may better
tolerate different intracellular or extracellular stresses
than primary cultures. Another possibility is that the
normal wt APP may be more readily metabolized than

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639

Fig. 8. Photomicrographs of hippocampal neurons treated with different adenoviral constructs

viewed by phase-contrast and fluorescent Sytox labeling. Cultures were prepared as described
in Materials and Methods and were infected with 10 pfu/cell of different adenoviral recombinants, as
shown. Phase-contrast views are of the same fields shown by fluorescence. Bar, 100 m.

Fig. 9. Percentage of dead cells in hippocampal cultures

infected with different adenoviral constructs using Sytox labeling.
Abbreviations are the same as those in Figure 7. Results are the
mean (SEM) of nine separate experiments, each containing at
least three individual evaluations. Asterisks represent a statistically
significant difference from null cultures (*P 0.05, **P 0.005).

the abnormal mutant APP in PC12 cells. The increased

amount of intracellular CTF immunoreactivity recognized by alpha-6 antibody in the AdxV642F-infected
cells supports this inference. Thus, primary hippocampal
neurons may be more vulnerable to the stresses resulting
from overexpression of h-APP than are the PC12 cells. As

discussed above, elevated intracellular levels of the

full-length APP in the infected cells may lead to more
CTF and/or A protein production, which may have toxic
effects on the cultured cells (Koh et al., 1990; Cotman et
al., 1992; Malouf, 1992; Busciglio et al., 1993; Jarrett et
al., 1993; Shearman et al., 1994; Simmons et al., 1994;
Kaneko et al., 1995; Harris et al., 1996; Itoh et al., 1996;
Sandhu et al., 1996; Blanc et al., 1997; Mark et al., 1997;
Lambert et al., 1998). Nishimura et al. (1998) reported
that injection of adenovirus overexpressing wt human
APP in rat hippocampus caused neuronal apoptosis. Their
results support our current observation on primary hippocampal neuronal death induced by overexpression of wt
h-APP. In primary cortical neuronal cultures, AdxS also
caused significant neuron death (manuscript in preparation).
Interestingly, a significant increase in the number of
the dead cells was also observed in primary hippocampal
cultures infected with AdxAS (Fig. 9). Because overexpression of antisense h-APPmRNA eliminates endogenous APP, a lack of normal APP function(s) may explain
these results. Accumulating evidence suggests that secreted forms of APP rather than membrane-associated
APP have trophic effects on PC12 cells and primary
cortical neurons (Araki et al., 1991; Yamamoto et al.,
1994; Wallace et al., 1997a). A recent report has demonstrated that there is a deficiency in the survival and
differentiation of hippocampal neurons cultured from
APP knockout mice (Perez et al., 1997). In support of
neurotrophic effects of APP, we showed that secreted APP

640

Luo et al.

was able to support PC12 cell, primary cortical, and

hippocampal neuronal survival (Luo, Kusiak and Wallace, manuscript in preparation). We found the APP effect
occurs, at least in part, through activation of the insulin
signaling pathway (Wallace et al., 1997b). Adenoviral
vector introducing antisense APP mRNA may mimic the
APP knockout situation and therefore abolish the normal
functions of APP, which may be necessary for neuron
survival in culture. In fact, in cultures of NGFdifferentiated PC12 cells, AdxAS treatment resulted in a
dramatic neurite retraction, although the cell bodies
remained apparently healthy (Luo, Kusiak and Wallace,
manuscript in preparation). No such effect was observed with AdxS or AdxNull. The results from antisense APP treatment in PC12 cell cultures are consistent with those of previous reports (LeBlanc et al.,
1992; Majocha et al., 1994; Allinquant et al., 1995).
Similar results with AdxAS were also observed in
primary cortical neuronal cultures (data not shown).
Thus, the mechanism causing primary neuronal cell
death induced by AdxAS may be different from that
induced by AdxS and AdxV642F. These results also
suggest that primary neuronal cultures may be a more
sensitive model in the evaluating cellular responses to
APP.
In summary, transfer of APP genes into cells by
adenoviral vectors is highly efficient and reproducible.
Overexpression of mutant APP induced cell death. The
present results suggest that adenoviral-mediated gene
transfer will be useful in the study of pathogenesis of AD
and in establishing a strategy of pharmacological intervention for AD.
ACKNOWLEDGMENTS
We thank Darrell D. Norton and Michael S. Prenger
for their technical support. This research was supported
by the Intramural Program, NIA, NIH, and a National
Research Council-NIA/NIH award to J.-J.L.
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